Conjugation of Streptococcal Capsular Saccharides

ABSTRACT

Three conjugation methods for use with the capsular saccharide of  Streptococcus agalactiae . In the first method, reductive amination of oxidized sialic acid residue side chains is used, but the aldehyde groups are first aminated, and then the amine is coupled to a carrier via a linker. In the second method, sialic acid residues and/or N-acetyl-glucosamine residues are de-N-acetylated to give amine groups, and the amine groups are coupled to a carrier protein via a linker. In the third method, linkage is via galactose residues in the capsular saccharide rather than sialic acid residues, which can conveniently be achieved using galactose oxidase.

All documents cited herein are incorporated by reference in theirentireties.

This application incorporates by reference the contents of a 117 kb textfile created on May 8, 2017 and named “00847600059sequencelisting.txt,”which is the sequence listing for this application.

TECHNICAL FIELD

This invention is in the field of conjugating bacterial capsularsaccharides to carriers in order to form glycoconjugates. Theglycoconjugates are useful for imunisation.

BACKGROUND ART

The capsular saccharides of bacteria have been used for many years invaccines against capsulated bacteria. As saccharides are T-independentantigens, however, they are poorly immunogenic. Conjugation to a carriercan convert T-independent antigens into T-dependent antigens, therebyenhancing memory responses and allowing protective immunity to develop.The most effective saccharide vaccines are therefore based onglycoconjugates, and the prototype conjugate vaccine was againstHaemophilus influenzae type b (‘Hib’) [e.g. see chapter 14 of ref 78].

Another bacterium for which conjugate vaccines have been described isStreptococcus agalactiae, also known as ‘group B streptococcus’, orsimply as ‘GBS’. Much of this work has been performed by Dennis Kasperand colleagues, and is described in documents such as references 1 to 9.The Kasper process for GBS saccharide conjugation typically involvesreductive amination of a purified saccharide to a carrier protein suchas tetanus toxoid (TT) or CRM197 [2]. The reductive amination involvesan amine group on the side chain of an amino acid in the carrier and analdehyde group in the saccharide. As GBS capsular saccharides do notinclude an aldehyde group in their natural form 20 then this isgenerated before conjugation by periodate oxidation of a portion of thesaccharide's sialic acid residues, as shown in FIG. 1 [2,10].

Although conjugate vaccines prepared in this manner for each of GBSserotypes Ia, Ib, II, III, and V have been shown to be safe andimmunogenic in humans [11], there remains a need for further and betterways of preparing conjugates of GBS capsular saccharides.

DISCLOSURE OF THE INVENTION

The invention is based on three conjugation methods that can be used inplace of the direct reductive amination disclosed in the prior art, allof which aim (a) to retain sialic acid residues in a form that is closerthan the prior art to the form see in the native polysaccharide, and (b)to allow the use of a linker in the conjugation reaction, in order toimprove coupling to carriers:

-   -   In the first method, reductive amination of oxidised sialic acid        residue side chains is used, but the aldehyde groups are first        aminated, and then the amine is coupled to a carrier via a        linker. This method is illustrated in ‘route A’ of FIG. 2.    -   In the second method, sialic acid residues and/or        N-acetyl-glucosamine residues are de-N-acetylated to give amine        groups, and the amine groups are coupled to a carrier protein        via a linker. This method is illustrated in ‘route B’ of FIG. 2.    -   In the third method, linkage is via galactose residues in the        capsular saccharide rather than sialic acid residues. This        method avoids disrupting key epitopes formed by sialic acid        residues.

In a first aspect, therefore, the invention provides a process forpreparing a conjugate of a S. agalactiae capsular saccharide and acarrier molecule, comprising the steps of: (a) oxidising a S. agalactiaecapsular saccharide in order to introduce an aldehyde group into atleast one terminal sialic acid residue in the saccharide; (b) subjectingthe aldehyde group to reductive amination with ammonia or a primaryamine, to give a —CH₂-linked amine; (c) reacting the —CH₂-linked aminewith a bifunctional linker, to give an activated saccharide; and (d)reacting the activated saccharide with a carrier molecule, therebygiving the conjugate. The invention also provides a conjugate, whereinthe conjugate comprises a S. agalactiae capsular saccharide moietyjoined to a carrier via a linker moiety, and wherein the linker moietyis attached to a sialic acid residue in the capsular saccharide moiety.

In a second aspect, the invention provides a process for preparing aconjugate of a S. agalactiae capsular saccharide and a carrier molecule,comprising the steps of: (a) de-N-acetylating the capsular saccharide,to give a de-N-acetylated saccharide; (b) reacting the de-N-acetylatedsaccharide with a bifunctional linker, to give an activated saccharide;and (c) reacting the activated saccharide with a carrier molecule,thereby giving the conjugate. Between steps (a) and (b), the process mayinvolve a step of partial re-N-acetylation of the saccharide.

In a third aspect, the invention provides a process for preparing aconjugate of a capsular saccharide and a carrier molecule, comprisingthe steps of: (a) oxidising a capsular saccharide in order to introducean aldehyde group into at least one galactose residue in the saccharide,to give a modified galactose residue; and (b) coupling the modifiedgalactose residue to a carrier molecule. The coupling in step (b) may bedirect, or may be via a linker molecule. The invention also provides aconjugate, wherein the conjugate comprises a capsular saccharide moietyjoined to a carrier via a linker moiety, and wherein the linker moietyis attached to a galactose residue in the capsular saccharide moiety.Oxidation of galactose residues is particularly useful for conjugationof S. agalactiae capsular saccharides, but is also suitable for use withother bacteria that have galactose-containing capsular saccharides e.g.in Neisseria meningitidis (serogroup W135), Vibrio cholerae (including0139), Klebsiella pneumoniae (including K21), Escherichia coli(including K52), Streptococcus pneumoniae (including type 18C), etc.This process can also be used with galactose-containinglipopolysaccharides and lipooligosaccharides. It is particularly usefulwhere the galactose is a terminal residue of the saccharide.

The Capsular Saccharide

The invention is based on the capsular saccharide of Streptococcusagalactiae. The capsular polysaccharide is covalently linked to thepeptidoglycan backbone of GBS, and is distinct from the group B antigen,which is another saccharide that is attached to the peptidoglycanbackbone.

The GBS capsular polysaccharides are chemically related, but areantigenically very different. All GBS capsular polysaccharides share thefollowing trisaccharide core:

β-D-GlcpNAc(1→3)β-D-Galp(1→4)β-D-Glcp

The various GBS serotypes differ by the way in which this core ismodified. The difference between serotypes Ia and III, for instance,arises from the use of either the GlcNAc (Ia) or the Gal (III) in thiscore for linking consecutive trisaccharide cores (FIG. 4). Serotypes Iaand Ib both have a [α-D-NeupNAc(2→3)β-D-Galp-(1→] disaccharide linked tothe GlcNAc in the core, but the linkage is either 1→4 (Ia) or 1→3 (Ib).

GBS-related disease arises primarily from serotypes Ia, Ib, II, III, IV,V, VI, VII, and VIII, with over 90% being caused by five serotypes: Ia,Ib, II, III & V. The invention preferably uses a saccharide from one ofthese five serotypes. As shown in FIG. 3, the capsular saccharides ofeach of these five serotypes include: (a) a terminal N-acetyl-neuraminicacid (NeuNAc) residue (commonly referred to as sialic acid), which inall cases is linked 2→3 to a galactose residue; and (b) aN-acetyl-glucosamine residue (GlcNAc) within the trisaccharide core.

All five saccharides include galactose residues within the trisaccharidecore, but serotypes Ia, Ib, II & III also contain additional galactoseresidues in each repeating unit, with the serotype II saccharidecontaining three galactose residues per repeating unit. In the thirdaspect of the invention, the galactose residues involved in theconjugation reactions may be a residue in the trisaccharide core or aresidue outside the trisaccharide core. Where a single saccharidemolecule is linked to multiple carrier molecules, it is preferred thatthe linkages involve the same-positioned galactose in the various linkedrepeating units, but it is also possible to link todifferently-positioned galactose residues in different repeating units.

Saccharides used according to the invention may be in their native form,or may have been modified. For example, the saccharide may be shorterthan the native capsular saccharide, or may be chemically modified.

Thus the saccharide used according to the invention may be asubstantially full-length capsular polysaccharide, as found in nature,or it may be shorter than the natural length. Full-lengthpolysaccharides may be depolymerised to give shorter fragments for usewith the invention e.g. by hydrolysis in mild acid, by heating, bysizing chromatography, etc. Chain length has been reported to affectimmunogenicity of GBS saccharides in rabbits [4]

Depolymerisation of the serotype III capsular saccharide byendo-β-galactosidase has been reported [refs. 1 & 4-6], including usingthe depolymerised material to form conjugates with a tetanus toxoidcarrier. Ozonolysis of capsular polysaccharides from GBS serotypes II,III and VIII has also been used for depolymerisation [12]. It ispreferred to use saccharides with MW>30 kDa, and substantiallyfull-length capsular polysaccharides can be used. For serotype Ia, it ispreferred to use polysaccharides with a MW up to ˜145 kDa. For serotypeIb, it is preferred to use polysaccharides with a MW up to ˜50 kDa. Forserotype III, it is preferred to use polysaccharides with a MW up to ˜50kDa. These molecular masses can be measured by gel filtration relativeto dextran standards, such as those available from Polymer StandardService [13].

The saccharide may be chemically modified relative to the capsularsaccharide as found in nature. For example, the saccharide may bede-O-acetylated (partially or fully), de-N-acetylated (partially orfully), N-propionated (partially or fully), etc. De-acetylation mayoccur before, during or after conjugation, but preferably occurs beforeconjugation. Depending on the particular saccharide, de-acetylation mayor may not affect immunogenicity e.g. the NeisVac-C™ vaccine uses ade-O-acetylated saccharide, whereas Menjugate™ is acetylated, but bothvaccines are effective. The relevance of O-acetylation on GBSsaccharides in various serotypes is discussed in reference 14, and it ispreferred to retain O-acetylation of sialic acid residues at positions7, 8 and/or 9 before during and after conjugation e.g. byprotection/de-protection, by re-acetylation, etc. The effect ofde-acetylation etc. can be assessed by routine assays.

Capsular saccharides can be purified by known techniques, as describedin the references herein. A typical process involves base extraction,centrifugation, filtration, RNase/DNase treatment, protease treatment,concentration, size exclusion chromatography, ultrafiltration, anionexchange chromatography, and further ultrafiltration. Treatment of GBScells with the enzyme mutanolysin, which cleaves the bacterial cell wallto free the cell wall components, is also useful.

As an alternative, the purification process described in reference 15can be used. This involves base extraction, ethanol/CaCl₂ treatment,CTAB precipitation, and re-solubilisation.

The invention is not limited to saccharides purified from naturalsources, however, and the saccharides may be obtained by other methods,such as total or partial synthesis.

The Carrier

The invention involves the use of carrier molecules, which are typicallyproteins. In general, covalent conjugation of saccharides to carriersenhances the immunogenicity of saccharides as it converts them fromT-independent antigens to T-dependent antigens, thus allowing primingfor immunological memory. Conjugation is particularly useful forpaediatric vaccines [e.g. ref. 16] and is a well known technique [e.g.reviewed in refs. 17 to 25].

Preferred carrier proteins are bacterial toxins or toxoids, such asdiphtheria toxoid or tetanus toxoid. The CRM197 mutant of diphtheriatoxin [26-28] is a particularly preferred carrier for, as is adiphtheria toxoid. Other suitable carrier proteins include the N.meningitidis outer membrane protein [29], synthetic peptides [30,31],heat shock proteins [32,33], pertussis proteins [34,35], cytokines [36],lymphokines [36], hormones [36], growth factors [36], human serumalbumin (preferably recombinant), artificial proteins comprisingmultiple human CD4⁺ T cell epitopes from various pathogen-derivedantigens [37] such as N19 [38], protein D from H. influenzae [39,40],pneumococcal surface protein PspA [41], pneumolysin [42], iron-uptakeproteins [43], toxin A or B from C. difficile [44], a GBS protein (seebelow; particularly GBS67) [195], etc.

Attachment to the carrier is preferably via a —NH₂ group e.g. in theside chain of a lysine residue in a carrier protein, or of an arginineresidue. Where a saccharide has a free aldehyde group then this canreact with an amine in the carrier to form a conjugate by reductiveamination. The third aspect of the invention may be based on reductiveamination involving an oxidised galactose in the saccharide (from whichan aldehyde is formed) and an amine in the carrier or in the linker.Attachment may also be via a —SH group e.g. in the side chain of acysteine residue.

It is possible to use more than one carrier protein e.g. to reduce therisk of carrier suppression. Thus different carrier proteins can be usedfor different GBS serotypes e.g. serotype Ia saccharides might beconjugated to CRM197 while serotype Ib saccharides might be conjugatedto tetanus toxoid. It is also possible to use more than one carrierprotein for a particular saccharide antigen e.g. serotype IIIsaccharides might be in two groups, with some conjugated to CRM197 andothers conjugated to tetanus toxoid. In general, however, it ispreferred to use the same carrier protein for all saccharides.

A single carrier protein might carry more than one saccharide antigen[45,46]. For example, a single carrier protein might have conjugated toit saccharides from serotypes Ia and Ib. To achieve this goal, differentsaccharides can be mixed prior to the conjugation reaction. In general,however, it is preferred to have separate conjugates for each serogroup,with the different saccharides being mixed after conjugation. Theseparate conjugates may be based on the same carrier.

Conjugates with a saccharide:protein ratio (w/w) of between 1:5 (i.e.excess protein) and 5:1 (i.e. excess saccharide) are preferred. Ratiosbetween 1:2 and 5:1 are preferred, as are ratios between 1:1.25 and1:2.5. Ratios between 1:1 and 4:1 are also preferred. With longersaccharide chains, a weight excess of saccharide is typical. As shown inthe examples, a weight ratio between 1:1 and 4:1, and particularly 1:1and 3:1, can readily be achieved. In general, the invention provides aconjugate, wherein the conjugate comprises a S. agalactiae capsularsaccharide moiety joined to a carrier, wherein the weight ratio ofsaccharide:carrier is at least 2:1.

Compositions may include a small amount of free carrier [47]. When agiven carrier protein is present in both free and conjugated form in acomposition of the invention, the unconjugated form is preferably nomore than 5% of the total amount of the carrier protein in thecomposition as a whole, and more preferably present at less than 2% byweight.

After conjugation, free and conjugated saccharides can be separated.There are many suitable methods, including hydrophobic chromatography,tangential ultrafiltration, diafiltration etc. [see also refs. 48 & 49,etc.].

Where the composition of the invention includes a depolymerisedoligosaccharide, it is preferred that depolymerisation precedesconjugation.

Introduction of Aldehyde Groups

The first aspect of the invention involves oxidation of sialic acid toform an aldehyde, and the third aspect involves oxidation of galactoseto form an aldehyde. The aldehyde can then be used for reactions such asreductive amination.

Oxidation of hydroxyls to give aldehydes can be achieved chemically orenzymatically. These reactions will typically take place in aqueousconditions.

Methods for oxidation of sialic acids in GBS saccharides in order tointroduce aldehyde groups for reductive amination are known in the art[e.g. ref. 50]. Typical reactions to produce aldehydes in sialic acidsinclude the use of periodate salts, and particularly meta-periodates(e.g. sodium or potassium meta-periodate e.g. NaIO₄), to oxidise vicinalhydroxides [10]. Periodate oxidation has been reported for at leastserogroups II [3,50], III [2] and V [50]. Other oxidation conditions canbe used e.g. use of osmium tetroxide, etc.

In the third aspect of the invention, the —OH that is oxidised ispreferably the primary —OH (i.e. not the secondary or anomeric —OHgroups), which is attached to C-6. Thus it is preferred to convertgalactose into galactohexodialose. This can conveniently be achievedusing a galactose oxidase enzyme, from any suitable source (e.g. fromFusarium fungi, or Dactylium dendroides). The enzyme can be used inrecombinant form, or purified from its native source. The galactoseoxidase enzyme has EC number 1.1.3.9, and is also known asD-Galactose:oxygen 6-oxidoreductase. The enzyme uses a copper ioncofactor and can be inhibited by cyanide, diethyldithiocarbamate, azideand hydroxylamine, so use of these reagents prior to oxidation ispreferably avoided. The pH optimum for the D. dendroides is aroundneutral, which is thus the preferred pH for oxidation. A product of theenzymatic reaction is H₂O₂ (reduced oxygen), and the concentration ofthis product can be controlled e.g. if its presence is damaging to thesaccharide.

For both sialic acid and galactose, therefore, the preferred oxidationreactions involve the terminal carbon atoms in the monosaccharides i.e.the highest-numbered carbons by standard nomenclature.

The proportion of monosaccharide subunits in a saccharide chain that areconverted to include an aldehyde group can be controlled by varyingreaction conditions. For example, reference 50 reports that controlledperiodate oxidation of serotype II GBS polysaccharide resulted in themodification of 7% of sialic acid residues as determined by gaschromatography-mass spectrometry analysis, with a higher percentagebeing seen for serotype V GBS polysaccharide. Reference 2 reports 25%conversion for serotype III. Preliminary studies of reaction conditions(e.g. time, temperature, concentrations, etc.) can be performed to findoptimum conditions for any desired end result.

In general, it is typical to introduce aldehyde groups into between 5%and 50% (e.g. 10-40%, preferably between 15%-30%; or between 5% and 20%)of the total sialic acid or galactose monosaccharide units within asaccharide. Higher percentages lead to saccharides that are moredifficult to handle, without any corresponding increase inimmunogenicity,

Reductive Amination

In the first aspect of the invention, reductive amination of the newaldehyde group is used to give a group for attachment of the linker.Reductive amination can also be used in the third aspect of theinvention after the aldehyde group has been produced, either to attach alinker or for direct linkage to a carrier. Reductive amination is astandard technique in organic chemistry, and has been used extensivelyin the production of conjugates of capsular saccharides for vaccine use.

In the first aspect, the reductive amination involves either ammonia ora primary amine (NH₂R). This can conveniently be achieved by using anammonium salt (e.g. ammonium chloride) in combination with anappropriate reducing agent (e.g. cyanoborohydrides, such as sodiumcyanoborohydride NaBH₃CN; borane-pyridine; sodium triacetoxyborohydride;borohydride exchange resin; etc.). The result of reductive amination isthat C-8 in the sialic acid carries —NHR rather than ═O. This group canthen be used for attachment of a bifunctional linker for conjugation.

In the third aspect, the oxidised galactose has an aldehyde group onC-6. This group can be coupled to a bifunctional linker by reductiveamination in the same way as described above i.e. involving ammonia or aprimary amine. As an alternative, reductive amination can be used tolink the aldehyde to a carrier directly, without use of a linker, byutilising an amine group on the carrier.

Reductive amination will generally be carried out in a polar proticsolvent, such as water or alcohol.

Bifunctional Linker

The first and second aspects of the invention (and, optionally, thethird aspect) involve the use of a bifunctional linker. A bifunctionallinker is used to provide a first group for coupling to an amine groupin the modified capsular saccharide and a second group for coupling tothe carrier (typically for coupling to an amine in the carrier).

The first group in the bifunctional linker is thus able to react withthe amine group (—NHR) on the modified sialic acid (or galactose)residue. This reaction will typically involve an electrophilicsubstitution of the amine's hydrogen. The second group in thebifunctional linker is able to react with the amine group on thecarrier. This reaction will again typically involve an electrophilicsubstitution of the amine. The invention can use both heterobifunctionallinkers and homobifunctional linkers.

Where the reactions with both the saccharide and the carrier involveamines then it is preferred to use a bifunctional linker of the formulaX-L-X, where: the two X groups are the same as each other and can reactwith the amines; and where L is a linking moiety in the linker. Apreferred X group is N-oxysuccinimide. L preferably has formula-L′-L²-L′-, where L′ is carbonyl. Preferred L² groups are straight chainalkyls with 1 to 10 carbon atoms (e.g. C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₃,C₉, C₁₀) e.g. —(CH₂)₄—. A preferred linker is thus adipic acidN-hydroxysuccinimide diester (SIDEA):

Other X groups are those which form esters when combined with HO-L-OH,such as norborane, p-nitrobenzoic acid, and sulfo-N-hydroxysuccinimide.

Further bifunctional linkers reactive with amines for use with theinvention include acryloyl halides (e.g. chloride) [54], haloacylhalides[55], disuccinimidyl glutarate, disuccinimidyl suberate, ethylene glycolbis[succinimidylsuccinate], etc.

The linker will generally be added in molar excess to modifiedsaccharide.

The linker/saccharide reaction will generally take place in an aproticsolvent (e.g. DMSO, ethanol acetate, etc.), as the linkers are typicallyinsoluble in water. Where water-soluble linkers are used, however, thena wider range of solvents is available, including protic solvents suchas water. Suitable linkers include sulphonated forms, such assulphonated SIDEA:

De-N-Acetylation and Re-N-Acetylation

The sialic acid residues in GBS capsular saccharides are N-acetylated,as are the glucosamine residues within the trisaccharide core. Whereasthe first aspect of the invention introduces amine groups in at C-8 ofthe sialic acid via an aldehyde intermediate, the second aspect of theinvention uses amine groups produced by de-N-acetylation of the sialicacid and/or N-acetyl-glucosamine residues. The reaction schemes foramines produced in this way are generally the same as described for thefirst aspect of the invention.

De-N-acetylation of GBS saccharides can conveniently be achieved bytreating the saccharide with a base. As GBS saccharides can be purifiedby a process involving base extraction [51] then de-N-acetylation may bea side-reaction during purification.

Because N-acetyl groups may be part of important epitopes in GBSsaccharides, complete de-N-acetylation may not be desirable, but thisprocess is difficult to control. If the extent of de-N-acetylation isgreater than desired, therefore, the invention may involve a step ofcontrolled re-N-acetylation. Re-N-acetylation can conveniently beperformed using a reagent such as acetic anhydride (CH₃CO)₂O e.g. in 5%ammonium bicarbonate [52]. Rather than use re-N-acetylation, however,the inventors have found that base extraction of the saccharide frombacteria can, if performed quickly enough without prolonged storage ofthe saccharide, give a saccharide with less than 25% de-N-acetylation.

The result of de-N-acetylation and optional re-N-acetylation is asaccharide in which at least 1 sialic acid residue or glucosamine isde-N-acetylated. Typically, at least 60% of the sialic acid residues andglucosamine residues in the GBS saccharide are N-acetylated e.g. ≧70%,≧75%, ≧80%, ≧85%, ≧90%, etc. The remaining de-N-acetylated groups (i.e.—NH₂ groups) can be used for conjugation in the same way as describedfor the first aspect of the invention, except that the —NH— in the finalconjugate will be derived from the original saccharide rather than beingadded during the conjugation reaction.

These de- and re-acetylation reactions can be performed in aqueousconditions.

In embodiments of the first and third aspects of the invention where thealdehyde is reductively aminated, it is preferred that the saccharide issubstantially totally re-N-acetylated prior to the reductive amination(preferably prior to oxidation of galactose in the third aspect), inorder to avoid the presence of free amine groups on sialic acids thatwould otherwise offer alternative linking groups to the aminatedaldehyde.

The Conjugate

Conjugates of the invention are formed by mixing the modified GBSsaccharide with the carrier under suitable reaction conditions. Ingeneral, two types of conjugate can be made, as shown in FIG. 5: (a) aconjugate where an individual saccharide is attached to a single carriere.g. through its reducing terminus; and (b) a conjugate where anindividual saccharide is attached to multiple carriers e.g. becauseseveral monosaccharide subunits are reactive. In both situations asingle carrier protein can link to multiple saccharide molecules becauseit can have multiple exposed lysine side chains.

Conjugates of type (b) are more typical in the present invention,because the modified sialic acid or galactose residues of the inventionoccur at multiple sites along a single saccharide [50]. In preferredconjugates of the invention, therefore, a single saccharide molecule iscoupled on average to more than one carrier molecule.

In the first and third methods of the invention, where oxidisedsaccharides are used for conjugation, the number of carrier moleculesattached to a saccharide will depend on the number of free aldehydegroups that are present. As mentioned above, it is preferred that 5-50%of sialic acid (first method) or galactose (third method) residues inthe saccharide are oxidised.

In the first and second aspects of the invention (and optionally in thethird) the conjugates will include a linker moiety. This linker moietyoriginates neither in the saccharide nor the carrier, but is a thirdmolecule used during conjugate preparation, and can readily bedistinguished from both the saccharide and carrier protein in a finalconjugate product.

The linker moiety may include atoms such as carbon, hydrogen, oxygenand/or nitrogen. Linkers that comprise carbon and hydrogen arepreferred, and linkers that further comprise oxygen and/or nitrogen arealso preferred. Linkers that include nitrogen atoms may include a carbonatom bonded to a nitrogen atom, which in turn is bonded to a secondcarbon atom (—C—N—C—). Linkers that include an oxygen atom preferablyinclude it as part of a carbonyl group. Linker moieties with a molecularweight of between 30-500 Da are preferred. Linkers containing twocarbonyl groups are preferred.

A particularly preferred linker moiety is —NH—C(O)—(CH₂)_(n)—C(O)—,wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The value of n ispreferably 4. The terminal —NH— in this linker is preferably attached toa carbon atom from the saccharide moiety. The terminal —C(O)— ispreferably attached to a nitrogen atom in an amino acid side chain inthe carrier. The preferred linker moiety can conveniently be introducedby a process involving: reductive amination of an aldehyde in anoxidised sialic acid; reaction of the resulting —NH₂ group with abifunctional linker that is a diester (e.g. a disuccinimidyl ester) of adioic acid (e.g. of adipic acid, HOOC—(CH₂)₄—COOH); and reductiveamination of the product (see FIG. 6 [53]).

Other chemistries that can be used to attach a linker to a terminal —NH₂in a saccharide, which may have been introduced (as in the first aspectof the invention) or may be part of a de-N-acetylated monosaccharideresidue (as in the second aspect of the invention), include:

-   -   acryloylation (e.g. by reaction with acryloyl chloride),        followed by Michael-type addition to either the ε-NH₂ of an        amino acid side chain or to a —SH of a cysteine side chain [54].        The resulting linker is —NH—C(O)—(CH₂)₂— (propionamido), as        shown in FIG. 8, or —C(O)—(CH₂)₂— if an existing —NH₂ takes part        in the reaction.    -   reaction with a haloacylhalide, followed by reaction with the        ε-NH₂ of an amino acid side chain or to a —SH of a cysteine side        chain [55]. The linker is —NH—C(O)—CH₂— (as shown in FIG. 9) or        —C(O)—CH₂—, depending on whether an existing or added —NH₂ takes        part in the reaction.

Another preferred linker moiety is —C(O)—(CH₂)_(n)—C(O)—, wherein n is1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The value of n is preferably 4. Oneterminal —C(O)— in this linker is preferably attached to a nitrogencarbon atom from the saccharide moiety, and the other terminal —C(O)— ispreferably attached to a nitrogen atom in an amino acid side chain inthe carrier. The preferred linker moiety can conveniently be introducedby a process involving: reaction of a —NH₂ group in a de-N-acetylatedmonosaccharide unit with a bifunctional linker that is a diester (e.g. adisuccinimidyl ester) of a dioic acid (e.g. of adipic acid,HOOC—(CH₂)₄—COOH); and reductive amination of the product (FIG. 7).

Other options include conjugating via hydroxyl groups in the saccharide.Hydroxyls can be activated (e.g. by CNBr or CDAP) and then subjected toconjugation.

Combinations of Conjugates and Other Antigens

As well as providing individual conjugates as described above, theinvention provides a composition comprising a conjugate of the inventionand one or more further antigens.

The further antigen(s) may comprise further conjugates of the invention,and so the invention provides a composition comprising more than oneconjugate of the invention. The further antigen(s) may be GBS saccharideconjugates prepared by methods other than those of the invention, and sothe invention provides a composition comprising a first GBS saccharideconjugate and a second GBS saccharide conjugate, wherein the firstconjugate is a conjugate of the invention and the second conjugate isnot a conjugate of the invention.

The different GBS conjugates may include different types of conjugatefrom the same GBS serotype and/or conjugates from different GBSserotypes. For example, the invention provides a composition comprisingconjugates from two or three of serotypes Ia, Ib and III. Thecomposition will be produced by preparing separate conjugates (e.g. adifferent conjugate for each serotype) and then combining theconjugates.

The further antigen(s) may comprise GBS amino acid sequences, as set outbelow.

The further antigen(s) may comprise antigens from non-GBS pathogens.Thus the compositions of the invention may further comprise one or morenon-GBS antigens, including additional bacterial, viral or parasiticantigens. These may be selected from the following:

-   -   a protein antigen from N. meningitidis serogroup B, such as        those in refs. 56 to 62, with protein ‘287’ (see below) and        derivatives (e.g. ‘AG287’) being particularly preferred.    -   an outer-membrane vesicle (OMV) preparation from N. meningitidis        serogroup B, such as those disclosed in refs. 63, 64, 65, 66        etc.    -   a saccharide antigen from N. meningitidis serogroup A, C, W135        and/or Y, such as the oligosaccharide disclosed in ref. 67 from        serogroup C or the oligosaccharides of ref. 68.    -   a saccharide antigen from Streptococcus pneumoniae [e.g. refs.        69-71; chapters 22 & 23 of ref. 78].    -   an antigen from hepatitis A virus, such as inactivated virus        [e.g. 72, 73; chapter 15 of ref. 78].    -   an antigen from hepatitis B virus, such as the surface and/or        core antigens [e.g. 73,74; chapter 16 of ref. 78].    -   an antigen from hepatitis C virus [e.g. 75].    -   an antigen from Bordetella pertussis, such as pertussis        holotoxin (PT) and filamentous haemagglutinin (FHA) from B.        pertussis, optionally also in combination with pertactin and/or        agglutinogens 2 and 3 [e.g. refs. 76 & 77; chapter 21 of ref.        78].    -   a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter        13 of ref. 78].    -   a tetanus antigen, such as a tetanus toxoid [e.g. chapter 27 of        ref. 78].    -   a saccharide antigen from Haemophilus influenzae B [e.g. chapter        14 of ref. 78]    -   an antigen from N. gonorrhoeae [e.g. 56, 57, 58].    -   an antigen from Chlamydia pneumoniae [e.g. 79, 80, 81, 82, 83,        84, 85].    -   an antigen from Chlamydia trachomatis [e.g. 86].    -   an antigen from Porphyromonas gingivalis [e.g. 87].    -   polio antigen(s) [e.g. 88, 89; chapter 24 of ref. 78] such as        IPV.    -   rabies antigen(s) [e.g. 90] such as lyophilised inactivated        virus [e.g. 91, RabAvert™].    -   measles, mumps and/or rubella antigens [e.g. chapters 19, 20 and        26 of ref. 78].    -   influenza antigen(s) [e.g. chapters 17 & 18 of ref. 78], such as        the haemagglutinin and/or neuraminidase surface proteins.    -   an antigen from Moraxella catarrhalis [e.g. 92].    -   an antigen from Streptococcus pyogenes (group A streptococcus)        [e.g. 93, 94, 95].    -   an antigen from Staphylococcus aureus [e.g. 96].

Where a saccharide or carbohydrate antigen is used, it is preferablyconjugated to a carrier in order to enhance immunogenicity. Conjugationof H. influenzae B, meningococcal and pneumococcal saccharide antigensis well known.

Toxic protein antigens may be detoxified where necessary (e.g.detoxification of pertussis toxin by chemical and/or genetic means[77]).

Where a diphtheria antigen is included in the composition it ispreferred also to include tetanus antigen and pertussis antigens.Similarly, where a tetanus antigen is included it is preferred also toinclude diphtheria and pertussis antigens. Similarly, where a pertussisantigen is included it is preferred also to include diphtheria andtetanus antigens.

Antigens may be adsorbed to an aluminium salt.

One type of preferred composition includes further antigens fromsexually-transmitted pathogens, such as: herpesvirus; N. gonorrhoeae;papillomavirus; C. trachomatis; etc. Another type of preferredcomposition includes further antigens that affect the elderly and/or theimmunocompromised, and so the GBS antigens of the invention can becombined with one or more antigens from the following non-GBS pathogens:influenza virus, Enterococcus faecalis, Staphylococcus aureus,Staphylococcus epidermis, Pseudomonas aeruginosa, Legionellapneumophila, Listeria monocytogenes, Neisseria meningitidis, andparainfluenza virus.

Antigens in the composition will typically be present at a concentrationof at least 1 μg/ml each. In general, the concentration of any givenantigen will be sufficient to elicit an immune response against thatantigen.

As an alternative to using proteins antigens in the composition of theinvention, nucleic acid encoding the antigen may be used [e.g. refs. 97to 105]. Protein components of the compositions of the invention maythus be replaced by nucleic acid (preferably DNA e.g. in the form of aplasmid) that encodes the protein.

In practical terms, there may be an upper limit to the number ofantigens included in compositions of the invention. The number ofantigens (including GBS antigens) in a composition of the invention maybe less than 20, less than 19, less than 18, less than 17, less than 16,less than 15, less than 14, less than 13, less than 12, less than 11,less than 10, less than 9, less than 8, less than 7, less than 6, lessthan 5, less than 4, or less than 3. The number of GBS antigens in acomposition of the invention may be less than 6, less than 5, or lessthan 4.

Pharmaceutical Compositions and Methods

The invention provides a pharmaceutical composition comprising (a) aconjugate of the invention and (b) a pharmaceutically acceptablecarrier. Typical ‘pharmaceutically acceptable carriers’ include anycarrier that does not itself induce the production of antibodies harmfulto the individual receiving the composition. Suitable carriers aretypically large, slowly metabolised macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, sucrose [106], trehalose [107], lactose,and lipid aggregates (such as oil droplets or liposomes). Such carriersare well known to those of ordinary skill in the art. The vaccines mayalso contain diluents, such as water, saline, glycerol, etc.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present. Sterilepyrogen-free, phosphate-buffered physiologic saline is a typicalcarrier. A thorough discussion of pharmaceutically acceptable excipientsis available in reference 108.

Compositions of the invention may be in aqueous form (i.e. solutions orsuspensions) or in a dried form (e.g. lyophilised). If a dried vaccineis used then it will be reconstituted into a liquid medium prior toinjection. Lyophilisation of conjugate vaccines is known in the art e.g.the Menjugate™ product is presented in lyophilised form, whereasNeisVac-C™ and Meningitec™ are presented in aqueous form. To stabiliseconjugates during lyophilisation, it may be preferred to include a sugaralcohol (e.g. mannitol) or a disaccharide (e.g. sucrose or trehalose)e.g. at between 1 mg/ml and 30 mg/ml (e.g. about 25 mg/ml) in thecomposition.

Compositions may be presented in vials, or they may be presented inready-filled syringes. The syringes may be supplied with or withoutneedles. A syringe will include a single dose of the composition,whereas a vial may include a single dose or multiple doses.

Aqueous compositions of the invention are also suitable forreconstituting other vaccines from a lyophilised form. Where acomposition of the invention is to be used for such extemporaneousreconstitution, the invention provides a kit, which may comprise twovials, or may comprise one ready-filled syringe and one vial, with thecontents of the syringe being used to reactivate the contents of thevial prior to injection.

Compositions of the invention may be packaged in unit dose form or inmultiple dose form. For multiple dose forms, vials are preferred topre-filled syringes. Effective dosage volumes can be routinelyestablished, but a typical human dose of the composition has a volume of0.5 ml e.g. for intramuscular injection.

The pH of the composition is preferably between 6 and 8, preferablyabout 7. Stable pH may be maintained by the use of a buffer. If acomposition comprises an aluminium hydroxide salt, it is preferred touse a histidine buffer [109]. The composition may be sterile and/orpyrogen-free. Compositions of the invention may be isotonic with respectto humans.

Compositions of the invention are immunogenic, and are more preferablyvaccine compositions. Vaccines according to the invention may either beprophylactic (i.e. to prevent infection) or therapeutic (i.e. to treatinfection), but will typically be prophylactic. Immunogenic compositionsused as vaccines comprise an immunologically effective amount ofantigen(s), as well as any other components, as needed. By‘immunologically effective amount’, it is meant that the administrationof that amount to an individual, either in a single dose or as part of aseries, is effective for treatment or prevention. This amount variesdepending upon the health and physical condition of the individual to betreated, age, the taxonomic group of individual to be treated (e.g.non-human primate, primate, etc.), the capacity of the individual'simmune system to synthesise antibodies, the degree of protectiondesired, the formulation of the vaccine, the treating doctor'sassessment of the medical situation, and other relevant factors. It isexpected that the amount will fall in a relatively broad range that canbe determined through routine trials.

Within each dose, the quantity of an individual saccharide antigen willgenerally be between 1-50 μs (measured as mass of saccharide) e.g. about1 μg, about 2.5 μg, about 4 μg, about 5 μg, or about 10 μg.

GBS affects various areas of the body and so the compositions of theinvention may be prepared in various forms. For example, thecompositions may be prepared as injectables, either as liquid solutionsor suspensions. The composition may be prepared for pulmonaryadministration e.g. as an inhaler, using a fine powder or a spray. Thecomposition may be prepared as a suppository or pessary. The compositionmay be prepared for nasal, aural or ocular administration e.g. as spray,drops, gel or powder [e.g. refs 110 & 111]. Success with nasaladministration of pneumococcal saccharides [112,113], Hib saccharides[114], MenC saccharides [115], and mixtures of Hib and MenC saccharideconjugates [116] has been reported.

Compositions of the invention may include an antimicrobial, particularlywhen packaged in multiple dose format.

Compositions of the invention may comprise detergent e.g. a Tween(polysorbate), such as Tween 80. Detergents are generally present at lowlevels e.g. <0.01%.

Compositions of the invention may include sodium salts (e.g. sodiumchloride) to give tonicity. A concentration of 10±2 mg/mlNaCl istypical.

Compositions of the invention will generally include a buffer. Aphosphate buffer is typical.

Compositions of the invention will generally be administered inconjunction with other immunoregulatory agents. In particular,compositions will usually include one or more adjuvants. Such adjuvantsinclude, but are not limited to:

A. Mineral-Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminium salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates),sulphates, etc. [e.g. see chapters 8 & 9 of ref. 117], or mixtures ofdifferent mineral compounds (e.g. a mixture of a phosphate and ahydroxide adjuvant, optionally with an excess of the phosphate), withthe compounds taking any suitable form (e.g. gel, crystalline,amorphous, etc.), and with adsorption to the salt(s) being preferred.The mineral containing compositions may also be formulated as a particleof metal salt [118].

Aluminum salts may be included in vaccines of the invention such thatthe dose of Al³⁺ is between 0.2 and 1.0 mg per dose.

A typical aluminium phosphate adjuvant is amorphous aluminiumhydroxyphosphate with PO₄/Al molar ratio between 0.84 and 0.92, includedat 0.6 mg Al³⁺/ml. Adsorption with a low dose of aluminium phosphate maybe used e.g. between 50 and 100 μg Al³⁺ per conjugate per dose. Where analuminium phosphate it used and it is desired not to adsorb an antigento the adjuvant, this is favoured by including free phosphate ions insolution (e.g. by the use of a phosphate buffer).

B. Oil Emulsions Oil emulsion compositions suitable for use as adjuvantsin the invention include squalene-water emulsions, such as MF59 (5%Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicronparticles using a microfluidizer) [Chapter 10 of ref. 117; see alsorefs. 119-121]. MF59 is used as the adjuvant in the FLUAD™ influenzavirus trivalent subunit vaccine.

Particularly preferred adjuvants for use in the compositions aresubmicron oil-in-water emulsions. Preferred submicron oil-in-wateremulsions for use herein are squalene/water emulsions optionallycontaining varying amounts of MTP-PE, such as a submicron oil-in-wateremulsion containing 4-5% w/v squalene, 0.25-1.0% w/v Tween 80(polyoxyelthylenesorbitan monooleate), and/or 0.25-1.0% Span 85(sorbitan trioleate), and, optionally,N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphophoryloxy)-ethylamine(MTP-PE). Submicron oil-in-water emulsions, methods of making the sameand immunostimulating agents, such as muramyl peptides, for use in thecompositions, are described in detail in references 119 & 122-123.Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA)may also be used as adjuvants in the invention.

C. Saponin Formulations [Chapter 22 of Ref 117]

Saponin formulations may also be used as adjuvants in the invention.Saponins are a heterologous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponins isolated from thebark of the Quillaia saponaria Molina tree have been widely studied asadjuvants. Saponin can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs.

Saponin compositions have been purified using HPLC and RP-HPLC. Specificpurified fractions using these techniques have been identified,including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, thesaponin is QS21. A method of production of QS21 is disclosed in ref.124. Saponin formulations may also comprise a sterol, such ascholesterol [125].

Combinations of saponins and cholesterols can be used to form uniqueparticles called immunostimulating complexs (ISCOMs) [chapter 23 of ref.117]. ISCOMs typically also include a phospholipid such asphosphatidylethanolamine or phosphatidylcholine. Any known saponin canbe used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA,QHA and QHC. ISCOMs are further described in refs. 125-127. Optionally,the ISCOMS may be devoid of additional detergent(s) [128].

A review of the development of saponin based adjuvants can be found inrefs. 129 & 130.

D. Virosomes and Virus-Like Particles

Virosomes and virus-like particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages,Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein p1). VLPs are discussed furtherin refs. 131-136. Virosomes are discussed further in, for example, ref.137

E. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as non-toxic derivatives of enterobacteriallipopolysaccharide (LPS), Lipid A derivatives, immunostimulatoryoligonucleotides and ADP-ribosylating toxins and detoxified derivativesthereof. Non-toxic derivatives of LPS include monophosphoryl lipid A(MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. Apreferred “small particle” form of 3 De-O-acylated monophosphoryl lipidA is disclosed in ref. 138. Such “small particles” of 3dMPL are smallenough to be sterile filtered through a 0.22 μm membrane [138]. Othernon-toxic LPS derivatives include monophosphoryl lipid A mimics, such asaminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [139,140].

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. OM-174 is described for example in refs. 141 & 142.

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention include nucleotide sequences containing a CpG motif (adinucleotide sequence containing an unmethylated cytosine linked by aphosphate bond to a guanosine). Double-stranded RNAs andoligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. References 143, 144 and 145 disclose possible analogsubstitutions e.g. replacement of guanosine with2′-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotidesis further discussed in refs. 146-151.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT [152]. The CpG sequence may be specific for inducing a Th1 immuneresponse, such as a CpG-A ODN, or it may be more specific for inducing aB cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed inrefs. 153-155. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. See,for example, refs. 152 & 156-158.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (E. coli heat labile enterotoxin “LT”), cholera(“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylatingtoxins as mucosal adjuvants is described in ref. 159 and as parenteraladjuvants in ref. 160. The toxin or toxoid is preferably in the form ofa holotoxin, comprising both A and B subunits. Preferably, the A subunitcontains a detoxifying mutation; preferably the B subunit is notmutated. Preferably, the adjuvant is a detoxified LT mutant such asLT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins anddetoxified derivatives thereof, particularly LT-K63 and LT-R72, asadjuvants can be found in refs. 161-168. Numerical reference for aminoacid substitutions is preferably based on the alignments of the A and Bsubunits of ADP-ribosylating toxins set forth in ref. 169, specificallyincorporated herein by reference in its entirety.

F. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5,IL-6, IL-7, IL-12 [170], etc.) [171], interferons (e.g. interferon-γ),macrophage colony stimulating factor, and tumor necrosis factor.

G. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres [172] or mucoadhesives such as cross-linked derivatives ofpoly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides and carboxymethylcellulose. Chitosan and derivativesthereof may also be used as adjuvants in the invention [173].

H. Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable andnon-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide-co-glycolide) are preferred, optionally treated to have anegatively-charged surface (e.g. with SDS) or a positively-chargedsurface (e.g. with a cationic detergent, such as CTAB).

I. Liposomes (Chapters 13 & 14 of Ref 117)

Examples of liposome formulations suitable for use as adjuvants aredescribed in refs. 174-176.

J. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters [177]. Such formulations furtherinclude polyoxyethylene sorbitan ester surfactants in combination withan octoxynol [178] as well as polyoxyethylene alkyl ethers or estersurfactants in combination with at least one additional non-ionicsurfactant such as an octoxynol [179]. Preferred polyoxyethylene ethersare selected from the following group: polyoxyethylene-9-lauryl ether(laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steorylether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether,and polyoxyethylene-23-lauryl ether.

K. Polyphosphazene (PCPP)

PCPP formulations are described, for example, in refs. 180 and 181.

L. Muramyl Peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), andN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

M. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in theinvention include Imiquamod and its homologues (e.g. “Resiquimod 3M”),described further in refs. 182 and 183.

N. Thiosemicarbazone Compounds.

Examples of thiosemicarbazone compounds, as well as methods offormulating, manufacturing, and screening for compounds all suitable foruse as adjuvants in the invention include those described in ref. 184.The thiosemicarbazones are particularly effective in the stimulation ofhuman peripheral blood mononuclear cells for the production ofcytokines, such as TNF-α.

O. Tryptanthrin Compounds.

Examples of tryptanthrin compounds, as well as methods of formulating,manufacturing, and screening for compounds all suitable for use asadjuvants in the invention include those described in ref. 185. Thetryptanthrin compounds are particularly effective in the stimulation ofhuman peripheral blood mononuclear cells for the production ofcytokines, such as TNF-α.

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above. For example, the followingcombinations may be used as adjuvant compositions in the invention: (1)a saponin and an oil-in-water emulsion [186]; (2) a saponin (e.g.QS21)+a non-toxic LPS derivative (e.g. 3dMPL) [187]; (3) a saponin (e.g.QS21)+a non-toxic LPS derivative (e.g. 3dMPL)+a cholesterol; (4) asaponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol) [188]; (5)combinations of 3dMPL with, for example, QS21 and/or oil-in-wateremulsions [189]; (6) SAF, containing 10% squalane, 0.4% Tween80™, 5%pluronic-block polymer L121, and thr-MDP, either microfluidized into asubmicron emulsion or vortexed to generate a larger particle sizeemulsion. (7) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing2% squalene, 0.2% Tween 80, and one or more bacterial cell wallcomponents from the group consisting of monophosphorylipid A (MPL),trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferablyMPL+CWS (Detox™); and (8) one or more mineral salts (such as an aluminumsalt)+a non-toxic derivative of LPS (such as 3dMPL).

Other substances that act as immunostimulating agents are disclosed inchapter 7 of ref. 117.

The use of aluminium salt adjuvants is particularly preferred, andantigens are generally adsorbed to such salts. The Menjugate™ andNeisVac™ conjugates use a hydroxide adjuvant, whereas Meningitec™ uses aphosphate adjuvant. It is possible in compositions of the invention toadsorb some antigens to an aluminium hydroxide but to have otherantigens in association with an aluminium phosphate. In general,however, it is preferred to use only a single salt e.g. a hydroxide or aphosphate, but not both. Not all conjugates need to be adsorbed i.e.some or all can be free in solution.

Methods of Treatment

The invention also provides a method for raising an immune response in amammal, comprising administering a pharmaceutical composition of theinvention to the mammal. The immune response is preferably protectiveand preferably involves antibodies. The method may raise a boosterresponse.

The mammal is preferably a human. Where the vaccine is for prophylacticuse, the human is preferably a child (e.g. a toddler or infant) or ateenager; where the vaccine is for therapeutic use, the human ispreferably an adult. A vaccine intended for children may also beadministered to adults e.g. to assess safety, dosage, immunogenicity,etc. A preferred class of humans for treatment are females ofchild-bearing age (e.g. teenagers and above). Another preferred class ispregnant females.

The invention also provides a composition of the invention for use as amedicament. The medicament is preferably able to raise an immuneresponse in a mammal (i.e. it is an immunogenic composition) and is morepreferably a vaccine.

The invention also provides the use of a conjugate of the invention inthe manufacture of a medicament for raising an immune response in amammal.

These uses and methods are preferably for the prevention and/ortreatment of a disease caused by S. agalactiae e.g. neonatal sepsis orbacteremia, neonatal pneumonia, neonatal meningitis, endometritis,osteomyelitis, septic arthritis, etc.

The subject in which disease is prevented may not be the same as thesubject that receives the conjugate of the invention. For instance, aconjugate may be administered to a female (before or during pregnancy)in order to protect offspring (so-called ‘maternal immunisation’[190-192]).

One way of checking efficacy of therapeutic treatment involvesmonitoring GBS infection after administration of the composition of theinvention. One way of checking efficacy of prophylactic treatmentinvolves monitoring immune responses against the GBS antigens afteradministration of the composition.

Preferred compositions of the invention can confer an antibody titre ina patient that is superior to the criterion for seroprotection for eachantigenic component for an acceptable percentage of human subjects.Antigens with an associated antibody titre above which a host isconsidered to be seroconverted against the antigen are well known, andsuch titres are published by organisations such as WHO. Preferably morethan 80% of a statistically significant sample of subjects isseroconverted, more preferably more than 90%, still more preferably morethan 93% and most preferably 96-100%.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or by rectal, oral, vaginal,topical, transdermal, intranasal, ocular, aural, pulmonary or othermucosal administration. Intramuscular administration to the thigh or theupper arm is preferred. Injection may be via a needle (e.g. a hypodermicneedle), but needle-free injection may alternatively be used. A typicalintramuscular dose is 0.5 ml.

The invention may be used to elicit systemic and/or mucosal immunity.

Dosage treatment can be a single dose schedule or a multiple doseschedule. Multiple doses may be used in a primary immunisation scheduleand/or in a booster immunisation schedule. A primary dose schedule maybe followed by a booster dose schedule. Suitable timing between primingdoses (e.g. between 4-16 weeks), and between priming and boosting, canbe routinely determined.

GBS Protein Antigens

As mentioned above, GBS proteins can be included in compositions of theinvention. These may be used as carrier proteins for conjugates of theinvention, carrier proteins for other conjugates, or as unconjugatedprotein antigens.

GBS protein antigens for use with the invention include those disclosedin references 93 and 193-195. Five preferred GBS protein antigens foruse with the invention are known as: GBS67; GBS80; GBS104; GBS276; andGBS322 [see ref. 93]. Further details of these five antigens are givenbelow.

The full-length sequences for these five GBS proteins are SEQ ID NOs 1to 5 herein. Compositions of the invention may thus include (a) apolypeptide comprising an amino acid sequence selected from SEQ ID NOs 1to 5, and/or (b) a polypeptide comprising (i) an amino acid sequencethat has sequence identity to one or more of SEQ ID NOs 1 to 5 and/or(ii) a fragment of SEQ ID NOs 1 to 5.

Depending on the particular SEQ ID NO, the degree of sequence identityin (i) is preferably greater than 50% (e.g. 60%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). Thesepolypeptides include homologs, orthologs, allelic variants andfunctional mutants. Typically, 50% identity or more between twopolypeptide sequences is considered to be an indication of functionalequivalence. Identity between polypeptides is preferably determined bythe Smith-Waterman homology search algorithm as implemented in theMPSRCH program (Oxford Molecular), using an affine gap search withparameters gap open penalty=12 and gap extension penalty=1.

Depending on the particular SEQ ID NO, the fragments of (ii) shouldcomprise at least n consecutive amino acids from the sequences and,depending on the particular sequence, n is 7 or more (e.g. 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100or more). The fragment may comprise at least one T-cell or, preferably,a B-cell epitope of the sequence. T- and B-cell epitopes can beidentified empirically (e.g. using PEPSCAN [196,197] or similarmethods), or they can be predicted (e.g. using the Jameson-Wolfantigenic index [198], matrix-based approaches [199], TEPITOPE [200],neural networks [201], OptiMer & EpiMer [202, 203], ADEPT [204], Tsites[205], hydrophilicity [206], antigenic index [207] or the methodsdisclosed in reference 208 etc.). Other preferred fragments are SEQ IDNOs 1 to 5 without their N-terminal amino acid residue or without theirN-terminal signal peptide. Removal of one or more domains, such as aleader or signal sequence region, a transmembrane region, a cytoplasmicregion or a cell wall anchoring motif can be used. Preferred fragmentsare given below (SEQ ID NOs 6 to 19).

These polypeptide may, compared to SEQ ID NOs 1 to 5, include one ormore (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conservative amino acidreplacements i.e. replacements of one amino acid with another which hasa related side chain. Genetically-encoded amino acids are generallydivided into four families: (1) acidic i.e. aspartate, glutamate; (2)basic i.e. lysine, arginine, histidine; (3) non-polar i.e. alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan; and (4) uncharged polar i.e. glycine, asparagine, glutamine,cystine, serine, threonine, tyrosine. Phenylalanine, tryptophan, andtyrosine are sometimes classified jointly as aromatic amino acids. Ingeneral, substitution of single amino acids within these families doesnot have a major effect on the biological activity. The polypeptides mayalso include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.)single amino acid deletions relative to SEQ ID NOs 1 to 5. Thepolypeptides may also include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8,9, 10, etc.) insertions (e.g. each of 1, 2, 3, 4 or 5 amino acids)relative to the SEQ ID NOs 1 to 5.

Polypeptides of the invention can be prepared in many ways e.g. bychemical synthesis (in whole or in part), by digesting longerpolypeptides using proteases, by translation from RNA, by purificationfrom cell culture (e.g. from recombinant expression), from the organismitself (e.g. after bacterial culture, or direct from patients), etc. Apreferred method for production of peptides <40 amino acids longinvolves in vitro chemical synthesis [209,210]. Solid-phase peptidesynthesis is particularly preferred, such as methods based on tBoc orFmoc [211] chemistry. Enzymatic synthesis [212] may also be used in partor in full. As an alternative to chemical synthesis, biologicalsynthesis may be used e.g. the polypeptides may be produced bytranslation. This may be carried out in vitro or in vivo. Biologicalmethods are in general restricted to the production of polypeptidesbased on L-amino acids, but manipulation of translation machinery (e.g.of aminoacyl tRNA molecules) can be used to allow the introduction ofD-amino acids (or of other non natural amino acids, such as iodotyrosineor methylphenylalanine, azidohomoalanine, etc.) [213]. Where D-aminoacids are included, however, it is preferred to use chemical synthesis.Polypeptides of the invention may have covalent modifications at theC-terminus and/or N-terminus.

If these GBS proteins are included in compositions of the invention thenthey can take various forms (e.g. native, fusions, glycosylated,non-glycosylated, lipidated, non-lipidated, phosphorylated,non-phosphorylated, myristoylated, non-myristoylated, monomeric,multimeric, particulate, denatured, etc.). They are preferably used inpurified or substantially purified form i.e. substantially free fromother polypeptides (e.g. free from naturally-occurring polypeptides),particularly from other GBS or host cell polypeptides).

GBS67

Nucleotide and amino acid sequence of GBS67 sequenced from serotype Vstrain 2603 V/R are set forth in ref. 93 as SEQ ID NOs 3745 & 3746. Theamino acid sequence is SEQ ID NO:1 herein:

MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQNKPLSKATFVLKTTAHPESKIEKVTAELTGEATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYEDTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEVGDLAHNKYKIELTVSGKTIVKPVDKQKPLDVVFVLDNSNSMNNDGPNFQRHNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKEDDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTDGVPTRSYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLFPLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINSLKQKNYDIFNEGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEITELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTIEDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILKGVKLEYIGNKLYVRGLNLGEGQKVILTYDVKLDDSFISNKFYDTNGRTTLNPKSEDPNTLRDFPIPKIRDVREYPTITIKNEKKLGEIEFIKVDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLIEAVSPEDYQKITNKPILTFEVVKGSIKNIIAVNKQISEYHEEGDKHLITNTHIPPKGIIPMTGGKGILSFILIGGAMMSIAGGIYIWKRYKKSSDMSIKK D

GBS67 contains a C-terminus transmembrane region which is indicated bythe underlined region closest to the C-terminus of SEQ ID NO: 1 above.One or more amino acids from the transmembrane region may be removed, orthe amino acid may be truncated before the transmembrane region. Anexample of such a GBS67 fragment is set forth below as SEQ ID NO: 18.

MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQNKPLSKATFVLKTTAHPESKIEKVTAELTGEATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYEDTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEVGDLAHNKYKIELTVSGKTIVKPVDKQKPLDVVFVLDNSNSMNNDGPNFQRHNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKEDDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTDGVPTRSYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLFPLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEITELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTIEDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILKGVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFISNKFYDTNGRTTLNPKSEDPNTLRDEPIPKIRDVREYPTITIKNEKKLGEIEFIKVDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLIEAVSPEDYQKITNKPILTFEVVKGSIKNIIAVNKQISEYHEEGDKHLITN THIPPKGIIPMTGGKGILS

GBS67 contains an amino acid motif indicative of a cell wall anchor,shown in italics in SEQ ID NO: 1 above. In some recombinant host cellsystems, it may be preferable to remove this motif to facilitatesecretion of a recombinant GBS67 protein from the host cell.Accordingly, in one preferred fragment of GBS67 for use in theinvention, the transmembrane and the cell wall anchor motif are removedfrom GBS67. An example of such a GBS67 fragment is set forth below asSEQ ID NO: 19.

MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQNKPLSKATFVLKTTAHPESKIEKVTAELTGEATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYEDTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEVGDLAHNKYKIELTVSGHTIVKPVDKQKPLDVVFVLDNSNSMNNDGPNFQRHNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKEDDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTDGVPTRSYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLFPLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEITELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTIEDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILKGVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFISNKFYDTNGRTTLNPKSEDPNTLRDFPIPKIRDVREYPTITIKNEKKLGEIEFIKVDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLIEAVSPEDYQKITNKPILTFEVVKGSIKNITAVNKQISEYHEEGDKHLITN THIPPKGI

GBS80

GBS80 refers to a putative cell wall surface anchor family protein.Nucleotide and amino acid sequence of GBS80 sequenced from serotype Visolated strain 2603 V/R are set forth in ref. 93 as SEQ ID NOs 8779 &8780. The amino acid sequence is set forth below as SEQ ID NO: 2:

MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTG GIGTAIFVAIGAAVMAFAVKGMKRR TKDN

GBS80 contains a N-terminal leader or signal sequence region which isindicated by the underlined sequence above. One or more amino acids fromthe leader or signal sequence region of GBS80 can be removed. An exampleof such a GBS80 fragment is set forth below as SEQ ID NO: 6:

AEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITEKPEKFKEIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTGGIGTAIFVAIGA AVMAFAVKGMKRRTKDN

GBS80 contains a C-terminal transmembrane region which is indicated bythe underlined sequence near the end of SEQ ID NO: 2 above. One or moreamino acids from the transmembrane region and/or a cytoplasmic regionmay be removed. An example of such a fragment is set forth below as SEQID NO:7:

MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTG

GBS80 contains an amino acid motif indicative of a cell wall anchor,shown in italics in SEQ ID NO: 2 above. In some recombinant host cellsystems, it may be preferable to remove this motif to facilitatesecretion of a recombinant GBS80 protein from the host cell. Thus thetransmembrane and/or cytoplasmic regions and the cell wall anchor motifmay be removed from GBS80. An example of such a fragment is set forthbelow as SEQ ID NO: 8.

MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTD ITVDSADATPDTIKNNKRPS

Alternatively, in some recombinant host cell systems, it may bepreferable to use the cell wall anchor motif to anchor the recombinantlyexpressed protein to the cell wall. The extracellular domain of theexpressed protein may be cleaved during purification or the recombinantprotein may be left attached to either inactivated host cells or cellmembranes in the final composition.

In one embodiment, the leader or signal sequence region, thetransmembrane and cytoplasmic regions and the cell wall anchor motif areremoved from the GBS80 sequence. An example of such a GBS80 fragment isset forth below as SEQ ID NO: 9:

AEVSQERPAKITVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHIDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPS

A particularly immunogenic fragment of GBS80 is located towards theN-terminus of the protein, and is given herein as SEQ ID NO: 10:

AEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQ NTLKITFKPEKFKEIAELLKG

GBS104

GBS104 refers to a putative cell wall surface anchor family protein. Ithas been referred to as einaA. Nucleotide and amino acid sequences ofGBS104 sequenced from serotype V isolated strain 2603 V/R are set forthin Ref. 93 as SEQ ID 8777 and SEQ ID 8778. The amino acid sequence isSEQ ID NO: 3 herein:

MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGEATFENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKENYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVVVLLDNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMGEMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTYDVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESLLGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTNLKADPNANKNQIGYLEGNGKHLITNTPKRPPGVFPKTGGIGTIVYILVGSTFMILTICSFRRKQL

GBS104 contains an N-terminal leader or signal sequence region which isindicated by the underlined sequence at the beginning of SEQ ID NO: 3above. One or more amino acids from the leader or signal sequence regionof GBS104 may be removed. An example of such a GBS104 fragment is setforth below as SEQ ID NO 11.

GETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGEATFENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKENYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVVVLLDNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMGEMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTYDVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESLLGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTNLKADPNANKNQIGYLEGNGKHLITNTPKRPPGVFPKTGGIGTIVYILVGSTFM ILTICSFRRKQL

GBS104 contains a C-terminal transmembrane and/or cytoplasmic regionwhich is indicated by the underlined region near the end of SEQ ID NO:3above. One or more amino acids from the transmembrane or cytoplasmicregions may be removed. An example of such a GBS104 fragment is setforth below as SEQ ID NO 12:

MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGEATFENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKENYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVVVLLDNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIRPKGYDIETVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMGEMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTYDVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESLLGAKFQLQIEKDFSGYKQEVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTNLKADPNANKNQIGYLEGNGKHLITN T

One or more amino acids from the leader or signal sequence region andone or more amino acids from the transmembrane or cytoplasmic regionsmay be removed. An example of such a GBS104 fragment is set forth belowas SEQ ID NO 13:

GETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGEATFENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKENYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVVVLLDNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMGEMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTYDVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESLLGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTNLKADPNANKNQIGYLEGNGKHLITNT

Further fragments of GBS104 include an 830 amino acid fragment of GBS104of amino acids 28-858 (numbered by SEQ ID NO: 3), a 359 amino acidfragment of GBS104 of amino acids 28-387, a 581 amino acid fragment ofGBS104 of amino acids 28-609, or a 740 amino acid fragment of GBS104 ofamino acids 28-768.

GBS276

GBS276 refers to a C5a peptidase. Further description of GBS276 can befound in references 214-217. Nucleotide and amino acid sequences ofGBS276 sequenced from serotype V isolated strain 2603 V/R are set forthin Ref. 93 as SEQ ID NOs 8941 & 8942. The amino acid sequence is SEQ IDNO: 4 herein:

MRKKQKLPFDKLAIALISTSILLNAQSDIKANTVTEDTPATEQAVEPPQPIAVSEESRSSKETKTSQTPSDVGETVADDANDLAPQAPAKTADTPATSKATIRDLNDPSHVKTLQEKAGKGAGTVVAVIDAGFDKNHEAWRLTDKIKARYQSKENLEKAKKEHGITYGEWVNDKVAYYHDYSKDGKNAVDQEHGTHVSGILSGNAPSEMKEPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRDAVNLGAKVINMSFGNAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKPRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQLTETATVKTDDHQDKEMPVISTNRFEPNKAYDYAYANRGTKEDDFKDVEGKIALIERGDIDFKDKIANAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISRRDGLLLKDNPPKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIKPDIAAPGQDILSSVANNKYAKLSGTSMSAPLVAGIMGLLQKQYETQYPDMTPSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGAGAVDAKKASAATMYVTDKDNTSSKVHLNNVSDKFEVTVTVHNKSDKPQELYYQVTVQTDKVDGKHFALAPKALYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPTKEELMSIPYIGFRGDFGNLSALEKPIYDSKDGSSYYHEANSDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEITETIFAGTFAKQDDDSHYYIHRHANGHPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGSTRFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDSRLTLASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNKPEQDGSDQAPDKKPEAKPEQDGSGQTPDKKKETKPEKDSSGQTPGKTPQKGQSSRTLEKRSSKRALATKASTRDQLPTTNDKDTNRLHLLKLVMTTFFLG

GBS276 contains an N-terminal leader or signal sequence region which isindicated by the underlined sequence at the beginning of SEQ ID NO: 4above. One or more amino acids from the leader or signal sequence regionof GBS276 may be removed. An example of such a GBS276 fragment is setforth below as SEQ ID NO: 14:

QSDIKANTVTEDTPATEQAVEPPQPIAVSEESRSSKETKTSQTPSDVGETVADDANDLAPQAPAKTADTPATSKATIRDLNDPSHVKTLQEKAGKGAGTVVAVIDAGFDKNHEAWRLTDKTKARYQSKENLEKAKKEHGITYGEWVNDKVAYYHDYSKDGKNAVDQEHGTHVSGILSGNAPSEMKEPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRDAVNLGAKVINMSFGNAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKPRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQLTETATVKTDDHQDKEMPVISTNRFEPNKAYDYAYANRGTKEDDFKDVEGKIALIERGDIDFKDKIANAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISRRDGLLLKDNPPKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIKPDIAAPGQDILSSVANNKYAKLSGTSMSAPLVAGIMGLLQKQYETQYPDMTPSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGAGAVDAKKASAATMYVTDKDNTSSKVHLNNVSDKFEVTVTVHNKSDKPQELYYQVTVQTDKVDGKHFALAPKALYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPTKEELMSIPYIGFRGDFGNLSALEKPIYDSKDGSSYYHEANSDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEITETIFAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGSTRFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDSRLTLASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNKPEQDGSDQAPDKKPEAKPEQDGSGQTPDKKKETKPEKDSSGQTPGKTPQKGQSSRTLEKRSSKRALATKASTRDQLPTTNDKDTNRLHLLK LVMTTFFLG

GBS276 contains a C-terminal transmembrane and/or cytoplasmic regionwhich is indicated by the underlined sequence near the end of SEQ ID NO:4 above. One or more amino acids from the transmembrane or cytoplasmicregions of GBS276 may be removed. An example of such a GBS276 fragmentis set forth below as SEQ ID NO: 15:

MRKKQKLPFDKLAIALISTSILLNAQSDIKANTVTEDTPATEQAVEPPQPIAVSEESRSSKETKTSQTPSDVGETVADDANDLAPQAPAKTADTPATSKATIRDLNDPSHVKTLQEKAGKGAGTVVAVIDAGFDKNHEAWRLTDKTKARYQSKENLEKAKKEHGITYGEWVNDKVAYYHDYSKDGKNAVDQEHGTHVSGILSGNAPSEMKEPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRDAVNLGAKVINMSFGNAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKPRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQLTETATVKTDDHQDKEMPVISTNRFEPNKAYDYAYANRGTKEDDFKDVEGKIALIERGDIDFKDKIANAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISRRDGLLLKDNPPKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIKPDIAAPGQDILSSVANNKYAKLSGTSMSAPLVAGIMGLLQKQYETQYPDMTPSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGAGAVDAKKASAATMYVTDKDNTSSKVHLNNVSDKFEVTVTVHNKSDKPQELYYQVTVQTDKVDGKHFALAPKALYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPTKEELMSIPYIGFRGDFGNLSALEKPIYDSKDGSSYYHEANSDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEITETIFAGTFAKQDDDSHYYIHRHANGHPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGSTRFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDSRLTLASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNKPEQDGSDQAPDKKPEAKPEQDGSGQTPDKKKETKPEKDSSGQTPGKTPQKGQSSRTLEKRSSKRAL ATK

One or more amino acids from the leader or signal sequence region andone or more amino acids from the transmembrane or cytoplasmic regions ofGBS276 may be removed. An example of such a GBS276 fragment is set forthbelow as SEQ ID NO: 16:

QSDIKANTVTEDTPATEQAVEPPQPIAVSEESRSSKETKTSQTPSDVGETVADDANDLAPQAPAKTADTPATSKATIRDLNDPSHVKTLQEKAGKGAGTVVAVIDAGFDKNHEAWRLIDKTKARYQSKENLEKAKKEHGITYGEWVNDKVAYYHDYSKDGKNAVDQEHGTHVSGILSGNAPSEMKEPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRDAVNLGAKVINMSFGNAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSEGGKPRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQLTETATVKTDDHQDKEMPVISTNRFEPNKAYDYAYANRGTKEDDFKDVEGKIALIERGDIDFKDKIANAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISRRDGLLLKDNPPKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIKPDIAAPGQDILSSVANNKYAKLSGTSMSAPLVAGIMGLLQKQYETQYPDMTPSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGAGAVDAKKASAATMYVTDKDNTSSKVELNNVSDKFEVTVTVHNKSDKPQELYYQVTVQTDKVDGKHFALAPKALYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLEGEVREKQDPTKEELMSIPYIGFRGDFGNLSALEKPTYDSKDGSSYYHEANSDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEITETIFAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGSTRFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDSRLTLASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNKPEQDGSDQAPDKKPEAKPEQDGSGQTPDKKKETKPEKDSSGQTPGKTPQKGQSSRTLEKRSSKRALATK

GBS322.

GBS322 refers to a surface immunogenic protein, also referred to as‘sip’. Nucleotide and amino acid sequences of GBS322 sequenced fromserotype V isolated strain 2603 V/R are set forth in Ref. 93 as SEQ IDNOs 8539 & 8540. The amino acid sequence is SEQ ID NO: 5 herein:

MNKKVLLTSTMAASLLSVASVQAQETDTTWTARTVSEVKADLVKQDNKSSYTVKYGDTLSVISEAMSIDMNVLAKINNIADINLIYPETTLTVTYDQKSHTATSMKIETPATNAAGQTTATVDLKTNQVSVADQKVSLNTISEGMTPEAATTIVSPMKTYSSAPALKSKEVLAQEQAVSQAAANEQVSPAPVKSITSEVPAAKEEVKPTQTSVSQSTTVSPASVAAETPAPVAKVAPVRTVAAPRVASVKVVTPKVETGASPEHVSAPAVPVTTTSPATDSKLQATEVKSVPVAQKAPTATPVAQPASTTNAVAAHPENAGLQPHVAAYKEKVASTYGVNEFSTYRAGDPGDHGKGLAVDFIVGTNQALGNKVAQYSTQNMAANNISYVIWQQKFYSNTNSIYGPANTWNAMPDRGGVTANHYDHVHVSFNK

GBS322 contains a N-terminal leader or signal sequence region which isindicated by the underlined sequence near the beginning of SEQ ID NO: 5.One or more amino acids from the leader or signal sequence region ofGBS322 may be removed. An example of such a GBS322 fragment is set forthbelow as SEQ ID NO: 17:

DLVKQDNKSSYTVKYGDTLSVISEAMSIDMNVLAKINNIADINLIYPETTLTVTYDQKSHTATSMKIETPATNAAGQTTATVDLKTNQVSVADQKVSLNTISEGMTPEAATTIVSPMKTYSSAPALKSKEVLAQEQAVSQAAANEQVSPAPVKSITSEVPAAKEEVKPTQTSVSQSTTVSPASVAAETPAPVAKVAPVRTVAAPRVASVKVVTPKVETGASPEHVSAPAVPVTTTSPATDSKLQATEVKSVPVAQKAPTATPVAQPASTTNAVAAHPENAGLQPHVAAYKEKVASTYGVNEFSTYRAGDPGDHGKGLAVDFIVGTNQALGNKVAQYSTQNMAANNISYVIWQQKFYSNTNSIYGPANTWNAMPDRGGVTANHYDHVHVSFNK

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example,x+10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

Where the invention provides a process involving multiple sequentialsteps, the invention can also provide a process involving less than thetotal number of steps. In the first aspect of the invention, forinstance, the invention provides a process comprising the steps of: (a)oxidising a GBS capsular saccharide in order to introduce an aldehydegroup into a terminal sialic acid residue; and (b) subjecting thealdehyde group to reductive amination. The further steps (c) and (d)need not be performed in order to fall within the scope of theinvention, as the product of steps (a) and (b) has utility as anintermediate in conjugate preparation, and may be used, stored,exported, etc. for separate and later use e.g. in steps (c) and (d).

Similarly, where a starting saccharide material is already partiallyprocessed then the invention encompasses processes involving only thelater steps of a method. In the third aspect of the invention, forinstance, the invention encompasses a process comprising a step ofcoupling a modified galactose residue to a carrier molecule, in whichthe starting material for the process is a saccharide that waspreviously oxidised to introduce an aldehyde group into a galactoseresidue.

These different steps can be performed at very different times bydifferent people in different places (e.g. in different countries).

It will be appreciated that sugar rings can exist in open and closedform and that, whilst closed forms are shown in structural formulaeherein, open forms are also encompassed by the invention. Similarly, itwill be appreciated that sugars can exist in pyranose and furanose formsand that, whilst pyranose forms are shown in structural formulae herein,furanose forms are also encompassed. Different anomeric forms of sugarsare also encompassed.

A primary amine can be represented by formula NH₂R. The R group willpreferably be electron donating, and includes C₁₋₈hydrocarbyl, morepreferably C₁₋₈alkyl, especially methyl. R is preferably —CH₃, —C₂H₅ or—C₃H₇. The hydrocarbyl may be substituted with one or more groups, suchas: halogen (e.g. Cl, Br, F, I), trihalomethyl, —NO₂, —CN,—N⁺(C₁₋₆alkyl)₂O; —SO₃H, —SOC₁₋₆alkyl, —SO₂C₁₋₆alkyl, —SO₃C₁₋₆alkyl,—OC(═O)OC₁₋₆alkyl, —C(═O)H, —C(═O)C₁₋₆alkyl, —OC(═O)C₁₋₆alkyl,—N(C₁₋₆alkyl)₂, C₁₋₆alkyl, —N(C₁₋₆alkyl)₂, —C(═O)N(C₁₋₆alkyl)₂,—N(C₁₋₆alkyl)C(═O)O(C₁₋₆alkyl), —N(C₁₋₆alkyl)C(═O)N(C₁₋₆alkyl)₂, —CO₂H,—OC(═O)N(C₁₋₆alkyl)₂, —N(C₁₋₆alkyl)C(═O)C₁₋₆alkyl,—N(C₁₋₆alkyl)C(═S)C₁₋₆alkyl, —N(C₁₋₆alkyl)SO₂N(C₁₋₆alkyl)₂,—CO₂C₁₋₆alkyl, —SO₂N(C₁₋₆alkyl)₂, —C(═O)NH₂, —C(═S)N(C₁₋₆alkyl)₂,—N(C₁₋₆alkyl) SO₂C₁₋₆alkyl, —N(C₁₋₆alkyl)C(═S)N(C₁₋₆alkyl)₂,—NH—C₁₋₆alkyl, —S—C₁₋₆alkyl or —O—C₁₋₆alkyl. The term ‘hydrocarbyl’includes linear, branched or cyclic monovalent groups consisting ofcarbon and hydrogen. Hydrocarbyl groups thus include alkyl, alkenyl andalkynyl groups, cycloalkyl (including polycycloalkyl), cycloalkenyl andaryl groups and combinations thereof, e.g. alkylcycloalkyl,alkylpolycycloalkyl, alkylaryl, alkenylaryl, cycloalkylaryl,cycloalkenylaryl, cycloalkylalkyl, polycycloalkylalkyl, arylalkyl,arylalkenyl, arylcycloalkyl and arylcycloalkenyl groups. Preferredhydrocarbyl are C₁₋₁₄ hydrocarbyl, more preferably C₁₋₈ hydrocarbyl.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows periodate oxidation of a terminal sialic acid residue.

FIG. 2 illustrates the first and second aspects of the invention.

FIG. 3 shows the repeating structures of capsular saccharides in GBSserotypes Ia, Ib, II, III & V.

FIG. 4 shows the difference between the repeating structures in GBSserotypes Ia and III.

FIG. 5 shows two types of conjugate that can be prepared.

FIG. 6 shows a preferred conjugation reaction using the succinimidyldiester of adipic acid, according to the first aspect of the invention.

FIG. 7 shows a preferred conjugation reaction using the succinimidyldiester of adipic acid, according to the second aspect of the invention.

FIGS. 8 and 9 shows the use of (8) acryloylation and (9) ahaloacylhalide, to prepare conjugates, after reductive amination of analdehyde formed by oxidation of a terminal sialic acid residue.

MODES FOR CARRYING OUT THE INVENTION Conjugate Production andCharacterisation

Capsular saccharide from GBS serotype Ib was purified as described inreference 15 and then re-acetylated as described above. The saccharidewas de-N-acetylated to provide amine groups for linking. These aminegroups were used to covalently conjugate the saccharides to monomerictetanus toxoid (TT) either by direct reductive amination (on C8 ofsialic acid, as described in the prior art) or via a SIDEA spacer (asdescribed for meningococcal saccharides in ref. 218).

Sialic acid content in the conjugates was determined was performedaccording to the colorimetric method of ref. 219. The total saccharideamount was extrapolated from sialic acid content (sialic acids are onaverage 31% by weight of the polymer). Protein concentration in theconjugate was determined with the Micro BCA Protein Assay Kit (Pierce).A polysaccharide:protein weight ratio of between 1 and 4 was the target,and results were as follows:

Saccharide Protein Conjugation (mg/ml) (mg/ml) Ratio Reductive animation1.740 1.271 1.37 SIDEA spacer 0.150 0.048 3.13

To investigate how the cross-linking ratio of conjugates could beaffected, purified GBS Ia and Ib saccharides were subjected to varyingdegrees of oxidation and then conjugated to CRM197. Results were asfollows

% Saccharide conc Protein conc Ratio oxidation (mg/ml) (mg/ml) (w/w) Ia5.0 1.188 0.468 2.54 14.2 1.360 0.776 1.75 44.7 1.018 0.690 1.48 79.02.989 2.012 1.49 86.0 1.737 1.074 1.62 Ib 4.3 2.544 1.437 1.77 12.02.821 2.383 1.18 46.7 3.644 3.941 0.92 79.6 3.821 3.770 1.01 80.2 1.2181.202 1.01

Similar experiments were used to study different protein carriers.CRM197 and tetanus toxoid were both used as carriers for GBS type IIIsaccharide, and results were:

% Saccharide conc Protein conc Ratio oxidation (mg/ml) (mg/ml) (w/w)CRM197 4.3 3.270 1.150 2.84 17.5 4.130 2.894 1.43 40.9 3.056 1.822 1.6861.8 3.165 2.358 1.34 78.9 4.230 4.502 0.94 Tetanus toxoid 3.9 1.0141.480 0.69 16.2 0.941 1.138 0.83 20.6 1.105 1.499 0.74 55.3 1.037 1.6000.65

Three different carriers were compared for GBS type II and Vsaccharides: tetanus toxoid; CRM197; and human serum albumin. The degreeof oxidation was 15.3% for the type V saccharide and 6.9% for the typeII saccharide. Results were:

Saccharide conc Protein conc Ratio (mg/ml) (mg/ml) (w/w) II 0.993 0.4442.24 2.999 1.541 1.95 2.146 0.890 2.41 V 1.308 0.902 1.45 1.272 0.8251.54 1.497 1.287 1.16

Human serum albumin was separately tested as a carrier for type Ia (6.7%oxidised), Ib (8.2% oxidised) and III (4.1% oxidised) saccharides:

Saccharide conc Protein conc Ratio Type (mg/ml) (mg/ml) (w/w) Ia 1.1120.784 1.42 Ib 3.710 3.078 1.21 III 3.318 2.869 1.16

Conjugates of type Ia, Ib and III were made using four differentcarriers: tetanus toxoid; CRM197; GBS80; and GBS67. With the tetanus andCRM carriers the % s oxidation were 9.1% for Ia, 14.2% for Ib and 13%for III; with the GBS carriers the % s were 8.2%, 9.0% and 7.9%. Animalsimmunised with the conjugates were then tested for protection againstthe respective GBS types (i.e. homologous challenge), and results wereas follows, expressed as the % of animals surviving lethal challenge:

PBS TT CRM197 GBS80 GBS67 control Ia 32 48 10 96 5 Ib 52 33 65 92 15 III76 60 71 82 0

In parallel experiments, with challenge by a GBS type V strain but noimmunisation with a type V saccharide, results were as follows:

PBS TT CRM197 GBS80 GBS67 control V 2 0 53 62 0

Thus the GBS carriers were able to provide some protection against thetype V strain, and so the use of GBS proteins as carriers offers abackground level of protein-mediated protection which can besupplemented by saccharides conjugated to the protein.

The level of free saccharide was tested for various conjugate lots, andresults were as follows:

GBS type Carrier free Ia CRM <1.0%  GBS80 3.5% GBS67  <1% Ib CRM 1.8%GBS80 14.8%  GBS67 <1.0%  III CRM 1.6% CRM 4.4% TetTox 3.8% GBS80 9.1%GBS67 <1.0% 

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES (THE CONTENTS OF WHICH ARE HEREBY INCORPORATED BY REFERENCE)

-   [1] Paoletti et al. (1990) J Biol. Chem 265:18278-83.-   [2] Wessels et al. (1990) J Clin Invest 86:1428-33.-   [3] Paoletti et al. (1992) Infect Immun 60:4009-14.-   [4] Paoletti et al. (1992) J Clin Invest 89:203-9.-   [5] Wessels et al. (1987) Proc Natl Acad Sci USA 84:9170-4.-   [6] Wang et al. (2003) Vaccine 21:1112-7. WO2006/082530    PCT/IB2006/000756-   [7] Wessels et al. (1993) Infect Immun 61:4760-6-   [8] Wessels et al. (1995) J Infect Dis 171:879-84.-   [9] Baker et al. (2004) J Infect Dis 189:1103-12.-   [10] U.S. Pat. No. 4,356,170.-   [11] Paoletti & Kasper (2003) Expert Opin Biol Ther 3:975-84.-   [12] U.S. Pat. No. 6,027,733 & 6274144.-   [13] www.polymer.de-   [14] Lewis et al. (2004) PNAS USA 101:11123-8.-   [15] International patent application PCT/IB2006/000626,    ‘PURIFICATION OF STREPTOCOCCAL CAPSULAR POLYSACCHARIDE’, claiming    priority from GB-0502096.1 (CHIRON SRL).-   [16] Ramsay et al. (2001) Lancet 357(9251):195-196.-   [17] Lindberg (1999) Vaccine 17 Suppl 2:S28-36.-   [18] Buttery & Moxon (2000) JR Coli Physicians Lond 34:163-168.-   [19] Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-33,    vii.-   [20] Goldblatt (1998) J. Med. Microbial. 47:563-567.-   [21] European patent 0477508.-   [22] U.S. Pat. No. 5,306,492.-   [23] WO98/42721.-   [24] Dick et al. in Conjugate Vaccines (eds. Cruse et al.) Karger,    Basel, 1989, 10:48-114.-   [25] Hermanson Bioconjugate Techniques, Academic Press, San    Diego (1996) ISBN: 0123423368.-   [26] Anonymous (January 2002) Research Disclosure, 453077.-   [27] Anderson (1983) Infect Immun 39(1):233-238.-   [28] Anderson et al. (1985) J Clin Invest 76(1):52-59.-   [29] EP-A-0372501.-   [30] EP-A-0378881.-   [31] EP-A-0427347.-   [32] WO93/17712-   [33] WO94/03208.-   [34] WO98/58668.-   [35] EP-A-0471177.-   [36] WO 91/01146-   [37] Falugi et al. (2001) Eur J Immunol 31:3816-24.-   [38] Baraldo et al. (2004) Infect Immun 72:4884-87.-   [39] EP-A-0594610.-   [40] WO00/56360.-   [41] WO02/091998.-   [42] Kuo et al. (1995) Infect Immun 63:2706-13.-   [43] WO01/72337-   [44] WO00/61761.-   [45] WO99/42130.-   [46] WO2004/011027.-   [47] WO96/40242.-   [48] Lei et al. (2000) Dev Biol (Basel) 103:259-264.-   [49] WO00/38711; U.S. Pat. No. 6,146,902.-   [50] WO94/06467.-   [51] U.S. Pat. No. 6,248,570.-   [52] Wessels et al. (1989) Infect Immun 57:1089-94.-   [53] U.S. Pat. No. 4,711,779.-   [54] WO00/10599.-   [55] U.S. Pat. No. 4,057,685.-   [56] WO99/24578.-   [57] WO99/36544.-   [58] WO99/57280.-   [59] WO00/22430.-   [60] Tettelin et al. (2000) Science 287:1809-1815.-   [61] WO96/29412.-   [62] Pizza et al. (2000) Science 287:1816-1820.-   [63] WO01/52885.-   [64] Bjune et al. (1991) Lancet 338(8775):1093-1096.-   [65] Fukasawa et al. (1999) Vaccine 17:2951-2958.-   [66] Rosenqvist et al. (1998) Dev. Biol. Stand. 92:323-333.-   [67] Costantino et al. (1992) Vaccine 10:691-698.-   [68] WO03/007985.-   [69] Watson (2000) Pediatr Infect Dis J 19:331-332.-   [70] Rubin (2000) Pediatr Clin North Am 47:269-285, v.-   [71] Jedrzejas (2001) Microbiol Mol Biol Rev 65:187-207.-   [72] Bell (2000) Pediatr Infect Dis J 19:1187-1188.-   [73] Iwarson (1995) APMIS 103:321-326.-   [74] Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80.-   [75] Hsu et al. (1999) Clin Liver Dis 3:901-915.-   [76] Gustafsson et al. (1996) N. Engl. J. Med. 334:349-355.-   [77] Rappuoli et al. (1991) TIBTECH 9:232-238.-   [78] Vaccines (2004) eds. Plotkin & Orenstein. ISBN 0-7216-9688-0.-   [79] WO02/02606.-   [80] Kalman et al. (1999) Nature Genetics 21:385-389.-   [81] Read et al (2000) Nucleic Acids Res 28:1397-406.-   [82] Shirai et al. (2000) J. Infect. Dis. 181(Suppl 3):S524-S527.-   [83] WO99/27105.-   [84] WO00/27994.-   [85] WO00/37494.-   [86] WO99/28475.-   [87] Ross et al. (2001) Vaccine 19:4135-4142.-   [88] Sutter et al. (2000) Pediatr Clin North Am 47:287-308.-   [89] Zimmerman & Spann (1999) Am Fam Physician 59:113-118, 125-126.-   [90] Dreesen (1997) Vaccine 15 Suppl:S2-6.-   [91] MMWR Morb Mortal Wkly Rep 1998 Jan. 16; 47(1):12, 19.-   [92] McMichael (2000) Vaccine 19 Suppl 1:S101-107.-   [93] WO02/34771.-   [94] Dale (1999) Infect Dis Clin North Am 13:227-43, viii.-   [95] Ferretti et al. (2001) PNAS USA 98: 4658-4663.-   [96] Kuroda et al. (2001) Lancet 357(9264):1225-1240; see also pages    1218-1219.-   [97] Robinson & Torres (1997) Seminars in Immunology 9:271-283.-   [98] Donnelly et al. (1997) Annu Rev Inmunol 15:617-648.-   [99] Scott-Taylor & Dalgleish (2000) Expert Opin Investig Drugs    9:471-480.-   [100] Apostolopoulos & Plebanski (2000) Curr Opin Mol Ther    2:441-447.-   [101] Ilan (1999) Curr Opin Mol Ther 1:116-120.-   [102] Dubensky et al. (2000) Mol Med 6:723-732.-   [103] Robinson & Pertmer (2000) Adv Virus Res 55:1-74.-   [104] Donnelly et al. (2000) Am J Respir Crit Care Med 162(4 Pt    2):S190-193.-   [105] Davis (1999) Mt. Sinai J. Med. 66:84-90.-   [106] Paoletti et al. (2001) Vaccine 19:2118-2126.-   [107] WO00/56365.-   [108] Gennaro (2000) Remington: The Science and Practice of    Pharmacy. 20th edition, ISBN: 0683306472.-   [109] WO03/009869.-   [110] Almeida & Alpar (1996) J. Drug Targeting 3:455-467.-   [111] Agarwal & Mishra (1999) Indian J Exp Biol 37:6-16.-   [112] WO00/53221.-   [113] Jakobsen et al. (2002) Infect Immun 70:1443-1452.-   [114] Bergquist et al. (1998) APMIS 106:800-806.-   [115] Baudner et al. (2002) Infect Immun 70:4785-4790.-   [116] Ugozzoli et al. (2002) J Infect Dis 186:1358-1361.-   [117] Vaccine Design (1995) eds. Powell & Newman. ISBN: 030644867X.    Plenum.-   [118] WO00/23105.-   [119] WO90/14837.-   [120] Podda (2001) Vaccine 19:2673-80.-   [121] Frey et al. (2003) Vaccine 21:4234-7.-   [122] U.S. Pat. No. 6,299,884.-   [123] U.S. Pat. No. 6,451,325.-   [124] U.S. Pat. No. 5,057,540.-   [125] WO96/33739.-   [126] EP-A-0109942.-   [127] WO96/11711.-   [128] WO00/07621.-   [129] Barr et al. (1998) Advanced Drug Delivery Reviews 32:247-271.-   [130] Sjolanderet et al. (1998) Advanced Drug Delivery Reviews    32:321-338.-   [131] Niikura et al. (2002) Virology 293:273-280.-   [132] Lenz et al. (2001) J Immunol 166:5346-5355.-   [133] Pinto et al. (2003) J Infect Dis 188:327-338.-   [134] Gerber et al. (2001) Virol 75:4752-4760.-   [135] WO03/024480-   [136] WO03/024481-   [137] Gluck et al. (2002) Vaccine 20:B10-B16.-   [138] EP-A-0689454.-   [139] Johnson et al. (1999) Bioorg Med Chem Lett 9:2273-2278.-   [140] Evans et al. (2003) Expert Rev Vaccines 2:219-229.-   [141] Meraldi et al. (2003) Vaccine 21:2485-2491.-   [142] Pajak et al. (2003) Vaccine 21:836-842.-   [143] Kandimalla et al. (2003) Nucleic Acids Research 31:2393-2400.-   [144] WO02/26757.-   [145] WO99/62923.-   [146] Krieg (2003) Nature Medicine 9:831-835.-   [147] McCluskie et al. (2002) FEMS Immunology and Medical    Microbiology 32:179-185.-   [148] WO98/40100.-   [149] U.S. Pat. No. 6,207,646.-   [150] U.S. Pat. No. 6,239,116.-   [151] U.S. Pat. No. 6,429,199.-   [152] Kandimalla et al. (2003) Biochemical Society Transactions 31    (part 3):654-658.-   [153] Blackwell et al. (2003) J Immunol 170:4061-4068.-   [154] Krieg (2002) Trends Immunol 23:64-65.-   [155] WO01/95935.-   [156] Kandimalla et al. (2003) BBRC 306:948-953.-   [157] Bhagat et al. (2003) BBRC 300:853-861.-   [158] WO03/035836.-   [159] WO95/17211.-   [160] WO98/42375.-   [161] Beignon et al. (2002) Infect Immun 70:3012-3019.-   [162] Pizza et al. (2001) Vaccine 19:2534-2541.-   [163] Pizza et al. (2000) Int J Med Microbiol 290:455-461.-   [164] Scharton-Kersten et al. (2000) Infect Inmmun 68:5306-5313.-   [165] Ryan et al. (1999) Infect Immun 67:6270-6280.-   [166] Partidos et al. (1999) Inmunol Lett 67:209-216.-   [167] Peppoloni et al. (2003) Expert Rev Vaccines 2:285-293.-   [168] Pine et al. (2002) J Control Release 85:263-270.-   [169] Domenighini et al. (1995) Mol Microbiol 15:1165-1167.-   [170] WO99/40936.-   [171] WO99/44636.-   [172] Singh et al] (2001) J Cont Release 70:267-276.-   [173] WO99/27960.-   [174] U.S. Pat. No. 6,090,406-   [175] U.S. Pat. No. 5,916,588-   [176] EP-A-0626169.-   [177] WO99/52549.-   [178] WO01/21207.-   [179] WO01/21152.-   [180] Andrianov et al. (1998) Biomaterials 19:109-115.-   [181] Payne et al. (1998) Adv Drug Delivery Review 31:185-196.-   [182] Stanley (2002) Clin Exp Dermatol 27:571-577.-   [183] Jones (2003) Curr Opin Investig Drugs 4:214-218.-   [184] WO04/60308-   [185] WO04/64759.-   [186] WO99/11241.-   [187] WO94/00153.-   [188] WO98/57659.-   [189] European patent applications 0835318, 0735898 and 0761231.-   [190] Glezen & Alpers (1999) Clin. Infect. Dis. 28:219-224-   [191] Madoff et al. (1994) J Clin Invest 94:286-92.-   [192] Paoletti et al. (1994) Infect Imnnun 62:3236-43.-   [193] WO03/093306.-   [194] WO2004/018646.-   [195] WO2004/041157.-   [196] Geysen et al. (1984) PNAS USA 81:3998-4002.-   [197] Carter (1994) Methods Mol Biol 36:207-23.-   [198] Jameson, B A et al. 1988, CABIOS 4(1):181-186.-   [199] Raddrizzani & Hammer (2000) BriefBioinform 1(2):179-89.-   [200] De Lalla et al. (1999) J. Immunol. 163:1725-29.-   [201] Brusic et al. (1998) Bioinformnatics 14(2):121-30-   [202] Meister et al. (1995) Vaccine 13(6):581-91.-   [203] Roberts et al. (1996) AIDS Res Hum Retroviruses 12(7):593-610.-   [204] Maksyutov & Zagrebelnaya (1993) Comput Appl Biosci 9(3):291-7.-   [205] Feller & de la Cruz (1991) Nature 349(6311):720-1.-   [206] Hopp (1993) Peptide Research 6:183-190.-   [207] Welling et al. (1985) FEBS Lett. 188:215-218.-   [208] Davenport et al. (1995) Immunogenetics 42:392-297.-   [209] Bodanszky (1993) Principles of Peptide Synthesis (ISBN:    0387564314).-   [210] Fields et al. (1997) Meth Enzymol 289: Solid-Phase Peptide    Synthesis. ISBN: 0121821900.-   [211] Chan & White (2000) Fmoc Solid Phase Peptide Synthesis. ISBN:    0199637245.-   [212] Kullmann (1987) Enzymatic Peptide Synthesis. ISBN: 0849368413.-   [213] Ibba (1996) Biotechnol Genet Eng Rev 13:197-216.-   [214] Qi Chen et al. (2002) Infect Immun 70:6409-15.-   [215] Beckmann et al. (2002) Infect Immun 70:2869-76.-   [216] Cheng et al. (2002) Infect Immun 70:2408-13.-   [217] Cheng et al. (2001) Infect Immun 69:2302-8.-   [218] WO03/007985.-   [219] Svennerholm (1958) Acta Chem. Scand. 12:547-554.

1. A process for preparing a conjugate of a Streptococcus agalactiaecapsular saccharide and a carrier molecule, comprising the steps of: (a)oxidising a S. agalactiae capsular saccharide in order to introduce analdehyde group into at least one terminal sialic acid residue in thesaccharide; (b) subjecting the aldehyde group to reductive aminationwith ammonia or a primary amine, to give a —CH₂-linked amine; (c)reacting the —CH₂-linked amine with a bifunctional linker, to give anactivated saccharide; and (d) reacting the activated saccharide with acarrier molecule, thereby giving the conjugate.
 2. A conjugatecomprising a Streptococcus agalactiae capsular saccharide moiety joinedto a carrier molecule via a linker moiety, wherein the linker moiety isattached to a sialic acid residue in the capsular saccharide moiety. 3.The process of claim 1, wherein the saccharide is from one of GBSserotypes Ia, Ib, II, III or V.
 4. The process of claim 1, wherein thesaccharide is selected from (a) a Streptococcus agalactiae capsularsaccharide having its native form; (b) a Streptococcus agalactiaecapsular saccharide that is shorter than its native form.
 5. The processof claim 1, wherein the saccharide is a chemically modifiedStreptococcus agalactiae capsular saccharide selected from (a) partiallyde-O-acetylated Streptococcus agalactiae capsular saccharide; (b) fullyde-O-acetylated Streptococcus agalactiae capsular saccharide; (c)partially de-N-acetylated Streptococcus agalactiae capsular saccharide;and (d) fully de-N-acetylated Streptococcus agalactiae capsularsaccharide.
 6. The process of claim 1, wherein the carrier molecule isselected from diphtheria toxoid, tetanus toxoid, CRM197, human serumalbumin, an artificial protein comprising multiple human CD4+ T cellepitopes from various pathogen-derived antigens, protein D from H.influenzae, or a S. agalactiae protein.
 7. The process of claim 1,wherein the carrier is attached to the saccharide via a —NH₂ group inthe carrier.
 8. The process of claim 1, wherein the conjugate has asaccharide:protein ratio (w/w) of between 1:5 and 5:1.
 9. The process ofclaim 1, wherein aldehyde groups are introduced into between 5% and 50%of the total sialic acid monosaccharide units.
 10. The process of claim1, wherein, after conjugation, free and conjugated saccharides areseparated.
 11. The process of claim 1, wherein reductive aminationinvolves either ammonia or a primary amine (NH₂R).
 12. The process ofclaim 17, wherein reductive amination involves an ammonium salt incombination with a reducing agent.
 13. The process of claim 1, whereinthe bifunctional linker is selected from heterobifunctional linkers andhomobifunctional linkers.
 14. The process of claim 20, where thereactions with both the saccharide and the carrier involve amines, andwherein the linker has formula X-L-X, where: the two X groups are thesame as each other and can react with the amines; and L is a linkingmoiety in the linker.
 15. The process of claim 21, wherein X isN-oxysuccinimide.
 16. The process of claim 22, wherein the linker isadipic acid N-hydroxysuccinimide diester.
 17. The process of claim 1,wherein the saccharide is substantially re-N-acetylated prior toreductive amination.
 18. The process of claim 1, wherein an individualsaccharide is attached to multiple carriers.
 19. A process for preparinga conjugate of a Streptococcus agalactiae capsular saccharide and acarrier molecule, comprising the steps of: (a) de-N-acetylating thecapsular saccharide, to give a de-N-acetylated saccharide; (b) reactingthe de-N-acetylated saccharide with a bifunctional linker, to give anactivated saccharide; and (c) reacting the activated saccharide with acarrier molecule, thereby giving the conjugate.
 20. A process forpreparing a conjugate of a capsular saccharide and a carrier molecule,comprising the steps of: (a) oxidising a capsular saccharide in order tointroduce an aldehyde group into at least one galactose residue in thesaccharide, to give a modified galactose residue; and (b) coupling themodified galactose residue to a carrier molecule.